NO341071B1 - Diarylthiohydantoin compounds - Google Patents

Description

The present invention relates to diarylhydantoin compounds including diarylthiohydantoins, and methods for synthesizing them and using them in the treatment of hormone-refractory prostate cancer.

Prostate cancer is the most common form of cancer and the second leading cause of cancer death in Western men. When the cancer is limited locally, the disease can be cured by surgery or radiation. However, 30% of such cancers recur with distant metastatic disease, and others have advanced disease at diagnosis. Advanced disease is treated by castration and/or administration of antiandrogens, the so-called trial androgen therapy. Castration reduces the circulating levels of androgens and reduces androgen receptor (AR) activity. Administration of antiandrogens blocks AR function by competing with androgen binding, which therefore reduces AR activity. Although initially effective, these treatments quickly fail and the cancer becomes hormone-refractory.

Recently, overexpression of AR has been identified and validated as a cause of hormone-refractory prostate cancer. See Chen, C.D., Welsbie, D.S., Tran, C, Baek, S.H., Chen, R, Vessella, R., Rosenfeld, M.G., and Sawyers, C.L., Molecular determinants of resistance to antiandrogen therapy, Nat. Med., 10: 33-39, 2004. Overexpression of AR is sufficient to cause progression from hormone-sensitive to hormone-refractory prostate cancer, suggesting that better AR inhibitors than current drugs may slow prostate cancer progression. It was demonstrated that AR and its ligand binding are required for the growth of hormone-refractory prostate cancer, indicating that AR remains a target for this disease. It was also demonstrated that overexpression of AR converts antiandrogens from antagonists to agonists in hormone-refractory prostate cancer (an AR antagonist inhibits AR activity, and an AR agonist stimulates AR activity). Data from this work explain why castration and antiandrogens fail to prevent prostate cancer progression and show unrecognized characteristics of hormone-refractory prostate cancer.

Bicalutamide (brand name: Casodex) is the most commonly used antiandrogen. While it has an inhibitory effect on AR in hormone-sensitive prostate cancer, it fails

in suppressing AR when cancer becomes hormone-refractory. Two weaknesses of current antiandrogens are blamed for the failure to prevent prostate cancer progression from the hormone-sensitive stage to the hormone-refractory disease and to effectively treat hormone-refractory prostate cancer. One is their weak antagonistic activities and the other is their strong agonistic activities when AR is overexpressed in hormone-

recalcitrant prostate cancer. Therefore, better AR inhibitors with more potent antagonistic activities and minimal agonistic activities are needed to delay disease progression and to treat the fatal hormone-refractory prostate cancer.

Non-steroidal antiandrogens, such as bicalutamide have been preferred over steroidal compounds for prostate cancer because they are more selective and have fewer side effects. This class of compounds has been described in many patents such as US Patent No. 4,097,578, US Pat. No. 5,411,981, US Pat. No. 5,705,654, PCT International Applications WO 97/00071 and WO 00/17163 and US Published Patent Application No. 2004/000996.

US Patent No. 5,434,176 includes broad claims encompassing a very large number of compounds, but synthetic routes are presented for only a small fraction of these compounds, and pharmacological data are presented for only two of them, and a person skilled in the art cannot easily imagine other specific connections.

Because the mechanism of hormone-refractory prostate cancer was not known, there was no biological system to test these compounds described in these patents for their effect on hormone-refractory prostate cancer. In particular, the possibility of AR overexpression in hormone-refractory prostate cancer to switch inhibitors from antagonists to agonists was not recognized. Some new features of hormone-refractory prostate cancer are reported in PCT applications US04/42221 and US05/05529. PCT International Application US05/05529 presented a methodology for identifying androgen receptor antagonist and agonist properties of compounds. However, for each compound produced, the time-consuming process of determining the antagonist and agonist properties of a compound must be determined. This means that there is no method to precisely predict properties relevant to treating prostate cancer from the chemical structure of a compound alone.

Some compounds have been reported to be inhibitors of the ligand binding domain (LBD) of the androgen receptor (AR). Several have been used as drugs to treat prostate cancer, e.g. bicalutamide (Casodex). Several binders of the AR LBD have been identified, e.g. the thiohydantoins RU59063 and BTID. (Teutsch, G.; Goubet, F.; Battmann, T.; Bonfils, A.; Bouchoux, F.; Cerede, E.; Gofflo, D.; Gaillard-Kelly, M.; Philibert. D. J. SteroidBoichem. Molec. Biol. 1994, 48, 111-119; Van Dort, M.E.; Robins, D.M.; Wayburn, B.J. Med. Chem. 2000, 43, 3344-3347).

FR 2 693 461 A describes compounds and their use as a medicament.

WO 2006/124118 A (NO 20076401) is only relevant in relation to novelty assessment and relates to diarylhydantoin compounds, which include diarylthiohydantoins, and methods for their synthesis and their use in the treatment of hormone-refractory prostate cancer.

There is a need for new thiohydantoin compounds having desirable pharmacological properties, and synthetic routes to prepare them. Because activities are sensitive to small structural changes, one compound may be effective in treating prostate cancer, while another compound may be ineffective, even if it differs from the first compound only slightly, as by the replacement of a single substituent.

Identification of compounds that have high potency to antagonize androgen activity, and that have minimal agonistic activity, should overcome hormone-refractory prostate cancer (HRPC) and avoid or slow the progression of hormone-sensitive prostate cancer (HSPC). Therefore, there is a need in the art for the identification of selective modulators of the androgen receptor, such as modulators that are non-steroidal, non-toxic and tissue-selective.

Summary of the invention

The invention provides a compound of formula

wherein R 1 and R 2 are, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms;

wherein R 3 is cyanoalkyl; and

where R 4 is hydrogen or fluorine.

The invention also provides a compound of formula

These compounds have strong antagonistic activities with minimal agonistic activities against AR. These compounds inhibit the growth of hormone-refractory prostate cancer.

Particular compounds of the invention include

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of the preceding compounds or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

The invention comprises such a pharmaceutical composition for use in the treatment of a hyperproliferative disorder. The hyperproliferative disorder may be hormone-refractory prostate cancer. The dosage can be in the range of from about 0.001 mg per kg body weight per day to around 100 mg per kg body weight per day, about 0.01 mg per kg body weight per day to around 100 mg per kg body weight per day, about 0.1 mg per kg body weight per day to about 10 mg per kg body weight per day, or about 1 mg per kg body weight per day.

The compound may be administered by intravenous injection, by injection into tissue, intraperitoneally, orally or nasally. The composition may have a form selected from the group consisting of a solution, dispersion, suspension, powder, capsule, tablet, pill, time-release capsule, time-release tablet and time-release pill.

The administered compound may be selected from the group consisting of NC56 or NC57, or a pharmaceutically acceptable salt thereof.

A method for synthesizing NC56 is described, comprising mixing NJf-dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile and DMF and heating under reflux to form a first mixture; and processing as above.

A method for synthesizing NC57 is described, comprising mixing DMSO, dichloromethane and oxalyl chloride to form a first mixture, adding 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo -6-thioxo-5,7-diazaspiro[3,4]octan-5-yl)phenyl)butanamide to the first mixture to form a second mixture; adding triethylamine to the second mixture to form a third mixture; heating the third mixture and quenching it with aqueous NH 4 Cl to form a fourth mixture; extracting an organic layer from the fourth mixture; and isolating the compound from the organic layer.

In one embodiment, a pharmaceutical composition comprises a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

The connection can e.g. have the formula

The pharmaceutical composition can e.g. have a form selected from the group consisting of a solution, dispersion, suspension, powder, capsule, tablet, pill, time-release capsule, time-release tablet and time-release pill. The compound may be administered by intravenous injection, by injection into tissue, intraperitoneally, orally or nasally. The composition may be administered at a dosage of the compound in the range of from about 0.001 mg per kg body weight per day to around 100 mg per kg body weight per day. The composition can be administered at a dosage of the compound in the range of from about 0.01 mg per kg body weight per day to around 100 mg per kg body weight per day. The composition can be administered at a dosage of the compound in the range of from about 0.1 mg per kg body weight per day to about 10 mg per kg body weight per day. The composition can be administered at a dosage of the compound of about 1 mg per kg body weight per day.

There is a method for treating prostate cancer comprising administering a pharmaceutical composition to an individual in need of such treatment, who thereby treats the prostate cancer. The pharmaceutical composition may interfere with the transcription of prostate-specific antigen mRNA. The pharmaceutical composition can prevent nuclear translocation of an androgen receptor protein. The pharmaceutical composition can destabilize an androgen receptor protein. The composition may be administered orally. The composition may have a form selected from the group consisting of a capsule, tablet and pill.

In one embodiment, the compound may be NC56 or NC57, a pharmaceutically acceptable salt of any of these, or combinations thereof.

A method for synthesizing compound [NC56] may include mixing iV,iV-dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile and DMF and heating under reflux to form a first mixture, adding an alcohol and an acid to the first mixture to form a second mixture, refluxing the second mixture, cooling the second mixture, combining the second mixture with water and extracting an organic layer, and isolating the compound from the organic made.

A method of synthesizing compound [NC57] may include mixing DMSO, dichloromethane and oxalyl chloride to form a first mixture, adding 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo -6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide to the first mixture to form a second mixture, add triethylamine to the second mixture to form a third mixture, heat the third the mixture and suppressing it with aqueous NH 4 Cl to form a fourth mixture, extracting an organic layer from the fourth mixture, isolating the compound from the organic layer.

A method described herein may include providing at least one diarylthiohydantoin compound, measuring inhibition of androgen receptor activity for the compound and determining whether the inhibition is above a first predetermined level, measuring stimulation of androgen receptor activity in hormone-refractory cancer cells for the compound and determining whether the stimulation is below a second at the predetermined level, selecting the compound if the inhibition is above the first predetermined level and the stimulation is below the second predetermined level. The predetermined levels may be those of bicalutamide. Measurement of inhibition may include measurement of inhibitory concentration (IC50) in an AR response reporter system or a prostate-specific antigen-secreting system. Measurement of stimulation may include measurement of the number of times induction at increasing concentrations in an AR response reporter system or a prostate-specific antigen-secreting system. Measuring inhibition and/or stimulation may include measuring an effect of the compound on tumor growth in an animal. The step of measuring inhibition and/or stimulation of androgen receptor activity may include measuring the binding affinity of an androgen receptor to the compound. The step of measuring inhibition and/or stimulation of androgen receptor activity may include measuring prevention of androgen receptor recruitment to at least one of prostate-specific antigen enhancer and prostate-specific antigen promoter. The step of measuring inhibition and/or stimulation of androgen receptor activity may include measuring prevention of androgen receptor nuclear translocation. The step of measuring inhibition and/or stimulation of androgen receptor activity may include measuring destabilization of an androgen receptor protein.

A method described herein may include contacting a mammalian cell capable of expressing prostate-specific antigen with a sufficient amount of a diarylthiohydantoin compound to interfere with the transcription of prostate-specific antigen mRMA. The diarylthiohydantoin compound may be selected from the group consisting of NC53, NC54, NC55, NC56 and NC57. The compound can prevent the formation of a transcription complex on a prostate-specific antigen. The compound can prevent an androgen receptor protein from complexing with a prostate-specific antigen gene. The compound can prevent an RNA polymerase II from complexing with a prostate-specific antigen gene.

One method includes contacting a mammalian cell with a sufficient amount of a diarylthiohydantoin compound to prevent nuclear translocation of an androgen receptor protein and/or to destabilize an androgen receptor protein.

The following figures present the results of pharmacological investigation of certain compounds. Figure 1 is a graph depicting that bicalutamide exhibits an agonistic effect on LNCaP-AR. Agonistic activities of bicalutamide in AR overexpressed hormone-refractory prostate cancer. LNCap cells with overexpressed AR were treated with increasing concentrations of DMSO as delivery vehicle or bicalutamide in the absence of RI 881. Activities of AR response reporters were measured. Figure 2 is a graph depicting an antagonistic test of bicalutamide on LNCaP-AR. Antagonistic activities of bicalutamide in hormone-refractory prostate cancer. LNCaP cells were treated with increasing concentrations of DMSO as delivery vehicle or bicalutamide in the absence of RI 881. AR response reporter activities were measured.

Figure 3 is a graph depicting the effect of compounds on LNCaP-AR.

Figure 4 a graph depicting the inhibition effect on LNCaP-AR.

Figure 5. Inhibitory effect on PSA expression of AR overexpressed LNCaP xenograft model. Mice were treated with delivery agent, 0.1, 1 or 10 mg per kg of example 7-3b (NC7) for 44 days orally once a day. The tumors were removed from the mice after 44 days of treatment, tumor lysate was extracted, and PSA level in tissue lysate was determined by ELISA. Figure 6 is a graph of tumor volume as a function of time for treatment with delivery vehicle solution, Casodex and NC53. Figure 7 is a graph of tumor size. AR overexpressing LNCaP cells were injected into the flanks of castrated SCID mice, subcutaneously. When the tumors reached about 100 cubic mm, they were randomized into five groups. Each group had nine animals. After they reached this tumor volume, they were orally given either delivery vehicle, bicalutamide, or NC53 at 10 or 50 mg/kg each day. The tumors were measured three-dimensionally, width, length and depth, by using a calibration target. Figure 8 depicts experimental results of tumor size. At day 18, the animals were imaged via an optical CCD camera, 3 hours after the last dose of treatment. An ROI was drawn over the tumor for luciferase activity measurement in photons/second. The right panels are a representation of the ROI measurements. Figure 9 is a graph depicting the pharmacokinetic curves of NC53 from intravenous (upper curve) and oral administration (lower curve). Figure 10 is a graph of fluorescence absorbance as a function of the logarithm of concentration, reflecting the binding affinities of several compounds to the rat androgen receptor. Figure 11 presents images reflecting the state of complex binding of androgen receptor and RNA polymerase II to the PSA enhancer and to the PSA promoter when Casodex or NC53 is added. Figure 12 presents images reflecting that androgen receptor translocates to the nucleus in the presence of Casodex, but not in the presence of NC53. Figure 13 presents images reflecting that androgen receptor is translocated to the nucleus in the presence of Casodex, but not in the presence of NC53. Figure 14 presents images reflecting that androgen receptor protein is degraded in the presence of NC53. Figure 15 is a diagram depicting prostate weight after treatment with various compounds. 10, 25 or 50 mg of compound per kilograms of body weight were administered per day, as indicated by the mark on the bar. The compounds were administered to healthy FVB mice. After treatment with compound for 14 days, the weight of the urogenital tract was determined by removing and weighing the semi-vesicles, prostate and bladder. Three mice were administered a given compound to obtain the data presented by a bar in the graph. One set of mice was not treated with a compound: data are presented in the bar labeled "untreated". Another set of mice was treated with delivery vehicle solution only: data are presented in the bar labeled "delivery vehicle". Figure 16 is a graph presenting a PSA test performed together with the experimental protocol presented in fig. 6. Figure 17 is a graph presenting the effect of different dose regimens of NC53 on tumor volume. Figure 18 is a graph presenting the rate of photon emission associated with luciferase activity at day 17 relative to the rate at day 0 after treatment with NC53 at doses of 0.1, 1 and 10 mg per kilogram of body weight per day and without treatment with NC53. Figure 19 presents the results of an experiment where SCID mice were injected with the LN-AR (HR) cell line to induce tumor growth. One set of mice was treated with the compound NC53 at a dose of 10 mg per kilogram of body weight per day; the other set of mice was treated with delivery vehicle solution only. (A) The relative tumor volume as a function of time shown for each set of mice. (B) Images of each set of mice with photon emission associated with luciferase activity at day 31 shown as color contours. (C) Rate of photon emission associated with luciferase activity shown at multiple time points for each set of mice. Figure 20 is a graph presenting PSA absorbance associated with LN-AR cells treated with various concentrations of NC53, NC54, NC55 and NC57 and delivery vehicle solution. Figure 21 is a graph presenting PSA absorbance associated with LN-CaP cells treated with various concentrations of NC7, NC48, NC53, bicalutamide and DMSO. Figure 22 presents results of an experiment performed with the wild-type non-transgenic mice (WT), castrated luciferase transgenic mice (Cast) and non-castrated luciferase transgenic mice (Intact). Data are shown for castrated luciferase transgenic mice treated with an implanted testosterone pellet that provided 12.5 mg per kilogram body weight with a 90 day release period (T/Cast), and data are shown for non-castrated luciferase transgenic mice treated with an implanted testosterone pellet that provided 12.5 mg per kilogram of body weight with a 90-day release period (Intact+T). Data are shown for castrated luciferase transgenic mice treated with the implanted testosterone pellet and with bicalutamide (BIC+T/Cast) or with NC53 (NC53+T/Cast) at 10 mg per kilogram of body weight per day. (A) Weight of urogenital tract at 14 days. (B) Photoemission rate at 14 days. In all cases, a hormone-refractory disease stage was not included. Figure 23 is a graph depicting PSA absorbance measured for LN-AR cells after treatment with various doses of several compounds. Figure 24 presents a table that provides several properties of compounds. Figure 15 also presents a graph providing the pharmacokinetic properties of several compounds in terms of serum concentration of compound as a function of time. Figure 25 is a graph of luciferase activity of the LIAR cell line dosed with various compounds administered at concentrations varying from 125 nmol to 1,000 nmol.

Detailed description

Embodiments of the invention are discussed in detail below. To describe embodiments, specific terminology is used for clarity. However, the invention is not intended to be limited to the specific terminology chosen.

Synthesis of diarylhydantoin compounds

Reference Example 56 [NC54JI] the following air- and moisture-sensitive reactions were carried out under an argon atmosphere using oven-dried glassware and standard syringe/septa techniques. The reactions were monitored with a SiC>2 TLC plate under UV light (254 nm) followed by visualization with a /?-anisaldehyde or ninhydrin color solution. Column chromatography was performed on silica gel 60. *H NMR spectra were measured at 400 MHz in CDCl3 unless otherwise stated, and data were reported as follows in ppm (8) from the internal standard (TMS, 0.0 ppm): chemical shift (multiplicity, integration, coupling constant in Hz).

Periodic acid (1.69 g, 7.41 mmol) was dissolved in acetonitrile (25 mL) with vigorous stirring, and then chromium trioxide (0.16 g, 1.60 mmol) was dissolved in the solution. 2-Fluoro-4-nitrotoluene (0.33 g, 2.13 mmol) was added to the above solution with stirring. A white precipitate was formed immediately with exothermic reaction. After stirring for 1 hour, the supernatant liquid of the reaction mixture was decanted into a bottle, and the solvent was removed by evaporation. The concentration residues were extracted with methylene chloride (2x30 ml) and water (2x30 ml). The organic layer was dried over MgSO4 and concentrated to give 2-fluoro-4-nitrobenzoic acid (formula 37) (0.32 mg, 81%) as a white solid.<*>H NMR 8 8.06 (ddd, 1 H, .7=9.9, 2.2 and 0.3), 8.13 (ddd, 1 H, .7=8.6, 2.2 and 0.9), 8.25 (ddd, 1 H, .7=8.6, 7.0 and 0.3).

Thionyl chloride (0.15 g, 1.30 mmol) was added slowly to a solution of 2-fluoro-4-nitrobenzoic acid (formula 37) (0.20 g, 1.10 mmol) in DMF (5 mL) cooled at - 5 °C. The mixture was stirred for an additional 1 hour at -5 °C. Excess methylamine (freshly distilled from its 40% aqueous solution) was added to the reaction medium. The second mixture was stirred for an additional 1 hour. Ethyl acetate (50 mL) was added to the mixture, which was washed with brine (2 x 50 mL). The organic layer was dried over MgSO 4 and concentrated to give N -methyl-2-fluoro-4-nitrobenzamide (Formula 38) (0.18 g, 85%) as a yellowish solid.<*>H NMR (acetone- ^) 8 3.05 (d, 3 H, .7=4.3), 6.31 (dd, 1 H, .7=13.5 and 2.1), 6.40 (dd, 1H, .7= 8.6 and 2.1), 7.64 (dd, 1H, J= 8.6 and 8.6).

A mixture of N-methyl-2-fluoro-4-nitrobenzamide (Formula 38) (0.18 g, 0.91 mmol) and iron (0.31 g, 5.60 mmol) in ethyl acetate (5 mL) and acetic acid (5 mL) was refluxed for 1 hour. The solid particles were filtered off. The filtrate was washed with water and extracted with ethyl acetate. The organic layer was dried over MgSO 4 , concentrated, and the residue was purified by SiO 2 column chromatography (dichloromethane:acetone, 95:5) to give N -methyl-2-fluoro-4-aminobenzamide (Formula 39) (0.14 g, 92 %) as an off-white solid.<*>H NMR (acetone-^) 6 2.86 (d, 3 H, .7=4.3), 5.50 (br s, 2 H), 6 .37 (dd, 1 H, .7=14.7 and 2.1), 6.50 (dd, 1H, .7=8.6 and 2.1), 7.06 (br s, 1H), 7.68 (dd, 1H, .7=8.8 and 8.8).

A mixture of N-methyl-2-fluoro-4-aminobenzamide (Formula 39) (96 mg, 0.57 mmol), acetone cyanohydrin (0.3 mL, 3.14 mmol) and magnesium sulfate (50 mg) was heated to 80 °C and stirred for 12 hours. Ethyl acetate (25 ml) was added to the medium and then washed with water (2 x 25 ml). The organic layer was dried over MgSO 4 and concentrated, and the residue was purified by SiO 2 column chromatography (dichloromethane:acetone, 95:5) to give N-methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide ( formula 40)

(101 mg, 75%) as a white solid.<*>H NMR δ 1.74 (s, 6 H), 2.98 (dd, 3 H, .7=4.8 and

1.1), 6.58 (dd, 1 H, .7=14.6 and 2.3), 6.63 (dd, 1 H, .7=8.7 and 2.3), 6.66 (br s, 1 H), 7.94 (dd, 1 H, .7=8.7 and 8.7).

4-Amino-2-trifluoromethylbenzonitrile (2.23 g, 12 mmol) was added portionwise over 15 min. to a well-stirred heterogeneous mixture of thiophosgene (1 mL, 13 mmol) in water (22 mL) at room temperature. Stirring was carried out for a further 1 hour. The reaction medium was extracted with chloroform (3x15 mL). The combined organic phase was dried over MgSO 4 and evaporated to dryness under reduced pressure to give the desired product 4-isothiocyanato-2-trifluoromethylbenzonitrile (formula 41) as a brownish solid and was used as is in the next step (2.72 g , 11.9 mmol, 99%).<*>H NMR δ 7.49 (dd, 1 H, .7=8.3 and 2.1), 7.59 (d, 1 H, .7=2 .1), 7.84 (d, 1 H, .7=8.3). 56-1) NC54 A mixture of N-methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide (formula 40) (30 mg, 0.13 mmol) and 4-isothiocyanato-2- Trifluoromethylbenzonitrile (Formula 41) (58 mg, 0.26 mmol) in DMF (1 mL) was heated under microwave irradiation at 100 °C for 11 h. To this mixture was added methanol (20 mL) and aqueous 1N HCl (5 mL). The second mixture was refluxed for 1.5 hours. After it was cooled to room temperature, the reaction mixture was poured into cold water (50 ml) and extracted with ethyl acetate (50 ml). The organic layer was dried over MgSO 4 , concentrated, and the residue was purified by SiO 2 column chromatography (dichloromethane:acetone, 95:5) to give NC54 (Formula 42) (15 mg, 25%) as a colorless crystal.<*>H NMR 8 1.61 (s, 6 H), 3.07 (d, 3 H, .7=4.1), 6.71 (m, 1 H), 7.15 (dd, 1H, .7=1 1.7 and 2.0), 7.24 (dd, 1H, .7=8.4 and 2.0), 7.83 (dd, 1H, .7=8.2 and 2.1), 7 .95 (d, 1H, .7=2.1), 7.99 (d, 1H, .7=8.2), 8.28 (dd, 1H, .7=8.4 and 8.4) .

Reference example 57

A mixture of N-methyl-2-fluoro-4-aminobenzamide (Formula 39) (62 mg, 0.37 mmol), cyclopentanone (0.07 mL, 0.74 mmol) and TMSCN (0.1 mL, 0, 74 mmol) was heated to 80 °C and stirred for 13 hours. Ethyl acetate (2 x 20 ml) was added to the medium and then washed with water (2 x 20 ml). The organic layer was dried over MgSO4 and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 95:5) to give N-methyl 2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide (Formula 43) (61 mg, 63%) as a white solid.<*>H NMR 6 7.95 (dd, 1H, J= 8.8, 8.8 Hz), 6.65 (br s, 1H), 6.59 (dd , 1H, J= 8.8, 2.3 Hz), 6.50 (dd, 1H, J= 14.6, 2.3 Hz), 4.60 (br s, 1H), 2.99 (dd , 3H, J= 4.8, 1.1 Hz), 2.36-2.45 (m, 2H), 2.10-2.18 (m, 2H), 1.82-1.95 (m , 4H).

57-1) NC55

A mixture of iN-Methyl 2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide (formula 43) (57 mg, 0.22 mmol) and 4-isothiocyanato-2-trifluoromethylbenzonitrile (0.15 g, 0.65 mmol) in DMF (3 mL) was heated under microwave irradiation (open vessel) at 130°C for 12 h. To this mixture was added methanol (20 mL) and 1N HCl (5 mL). The second mixture was refluxed for 1.5 hours. After it was cooled to room temperature, the reaction mixture was poured into cold water (50 ml) and extracted with ethyl acetate (50 ml). The organic layer was dried over MgSO4, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 95:5) to give 4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-4 -oxo-2-thioxo-1,3-diazaspiro[4,4]nonan-1-yl)-2-fluoro-1N-methylbenzamide, NC55 (Formula 44) (8 mg, 7%) as a pale yellowish solid .<*>H NMR 6 8.28 (dd, 1H, J= 8.4, 8.4 Hz), 7.98 (d, 1H, J = 8.3 Hz), 7.96 (d, 1H , J= 1.8 Hz), 7.84 (dd, 1H, J= 8.3, 1.8 Hz), 7.27 (dd, 1H, J= 8.4, 1.8 Hz), 7 .17 (dd, 1H, J= 11.7, 1.8 Hz), 6.67-6.77 (m, 1H), 3.07 (d, 3H, J= 4.3 Hz), 2, 32-2.41 (m, 2H), 2.13-2.21 (m, 2H), 1.85-1.96 (m, 2H), 1.49-1.59 (m, 2H).

Example 58

Trifluoroacetic anhydride (0.85 mL, 6.14 mmol) was added to a solution of 4-(4-aminophenyl)butyric acid (0.5 g, 2.79 mmol) in chloroform (10 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 3 hours. The mixture was partitioned with chloroform (20 mL) and water (20 mL). The organic layer was dried over MgSO4 , concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanoic acid (formula 45) (0.53 g, 69%).<*>H NMR δ 7.81 (br s, 1H), 7.48 (d, 2H, J= 8.5 Hz), 7.22 (d, 2H, J= 8.5 Hz), 2.68 (t, 2H, J= 7.5 Hz), 2.38 (t, 2H, J= 7.5 Hz), 1.96 (p, 2H, J = 7.5 Hz).

Thionyl chloride (71 mg, 0.60 mmol) was added slowly to a solution of 4-[4-(2,2,2-Trifluoroacetylamino)phenyl]butanoic acid (Formula 45) (0.15 g, 0.55 mmol) in DMF (5 mL) cooled at -5 °C. The mixture was stirred for an additional 1 hour at -5 °C. Excess dimethylamine (freshly distilled from its 40% aqueous solution) was added to the reaction medium. The second mixture was stirred for an additional 1 hour. Ethyl acetate (50 mL) was added to the mixture, which was washed with brine (2 x 50 mL). The organic layer was dried over MgSO4 and concentrated to give iV,iV-dimethyl 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanamide (formula 46) (0.17 g, quant.) as a yellowish solid. *H NMR δ 9.70 (br s, 1H), 7.55 (d, 2H, J= 8.6 Hz), 7.11 (d, 2H, J= 8.6 Hz), 2.91 ( s, 3H), 2.89 (s, 3H), 2.60 (t, 2H, J= 7.7 Hz), 2.27 (t, 2H, J= 7.7 Hz), 1.89 ( p, 2H, J= 7.7 Hz).

1 N NaOH solution (3 mL) was added to a solution of AUV-dimethyl 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanamide (Formula 46) (0.17 g, 0.55 mmol) in methanol (2 mL) at room temperature. The mixture was stirred for 14 hours. The mixture was partitioned with chloroform (25 mL) and water (25 mL). The organic layer was dried over MgSO4 and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give iV,iV-dimethyl 4-(4-aminophenyl)butanamide (formula 47) (74 mg, 66%) as a white solid.<*>H NMR δ 6.97 (d, 2H, J= 8.3 Hz), 6.61 (d, 2H, J = 8.3 Hz), 3 .56 (br s, 2H), 2.92 (s, 6 H), 2.56 (t, 2H, J= 7.7 Hz), 2.28 (t, 2H, J= 7.7 Hz) , 1.91 (p, 2H, J = 7.7 Hz).

A mixture of N^V-dimethyl 4-(4-aminophenyl)butanamide (Formula 47) (74 mg, 0.36 mmol), cyclobutanone (54 mg, 0.78 mmol) and TMSCN (77 mg, 0.78 mmol ) was heated to 80 °C and stirred for 15 h. Ethyl acetate (2 x 20 ml) was added to the medium and then washed with water (2 x 20 ml). The organic layer was dried over MgSO4 and concentrated, and the concentration residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give jV,iV-Dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide ( formula 48) (58 mg, 57%) as a white solid.<*>H NMR 8 7.07 (d, 2H, J= 8.5 Hz), 6.59 (d, 2H, J= 8, 5 Hz), 3.94 (br s, 1H), 2.94 (s, 3H), 2.93 (s, 3H), 2.75-2.83 (m, 2H), 2.60 (t , 2H, J= 7.6 Hz), 2.33-2.42 (m, 2H), 2.30 (t, 2H, J= 7.6 Hz), 2.11-2.28 (m, 2H), 1.93 (p, 2H, J = 7.6 Hz).

A mixture of N-N-dimethyl 4-[4-(l-cyanocyclobutylamino)phenyl]butanamide (formula 48) (58 mg, 0.20 mmol) and 4-isothiocyanato-2-trifluoromethylbenzonitrile (74 mg, 0.32 mmol) in DMF (3 mL) was heated under reflux for 2 h. To this mixture was added methanol (20 mL) and 1N HCl (5 mL). The second mixture was refluxed for 1.5 hours. After it was cooled to room temperature, the reaction mixture was poured into cold water (50 ml) and extracted with acetyl acetate (50 ml). The organic layer was dried over MgSO4, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 95:5) to give

4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)-iV,iV- dimethylbutanamide, NC56 (formula 49) (44 mg, 42%) as a faint yellowish solid.<*>H NMR δ 7.98 (s, 1H), 7.97 (d, 1H, J= 8.2 Hz ), 7.86 (d, 1H, J= 8.2 Hz), 7.42 (d, 2H, J= 8.3 Hz), 7.22 (d, 2H, J= 8.3 Hz), 2.99 (s, 3H), 2.96 (s, 3H), 2.78 (t, 2H, J= 7.5 Hz), 2.62-2.70 (m, 2H), 2.52 -2.63 (m, 2H), 2.40 (t, 2H, J= 7.5 Hz), 2.15-2.30 (m, 1H), 2.04 (p, 2H, J= 7 .5 Hz), 1.62-1.73 (m, 1H).

Reference example 59

A mixture of 4-(4-aminophenyl)butyric acid (0.20 g, 1.12 mmol), cyclobutanone (0.17 mL, 2.23 mmol) and TMSCN (0.30 mL, 2.23 mmol) was heated up to 80 °C and stirred for 13 hours. Ethyl acetate (2x30 ml) was added to the medium and then washed with water (2x30 ml). The organic layer was dried over MgSO4 and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give 4-[4-(1-cyanocyclobutylamino)phenyl]butanoic acid (formula 50) (0 .21 g, 74%) as a yellowish solid.<*>H NMR 8 7.06 (d, 2H, J= 8.6 Hz), 6.59 (d, 2H, J= 8.6 Hz) , 2.75-2.83 (m, 2H), 2.59 (t, 2H, J= 7.5 Hz), 2.37 (t, 2H, J= 7.5Hz), 2.33-2.42 (m, 2H), 2.11-2.28 (m, 2H), 1.92 (p, 2H, J = 7.5 Hz).

A mixture of 4-[4-(1-cyanocyclobutylamino)phenyl]butanoic acid (formula 50) (0.21 g, 0.83 mmol) and 4-isothiocyanato-2-trifluorobenzonitrile (0.25 g, 1.08 mmol) in toluene (10 mL) was heated under reflux for 1 h. To this mixture was added aqueous 1N HCl (5 mL). The second mixture was refluxed for 1.5 hours. After it was cooled to room temperature, the reaction mixture was poured into cold water (50 ml) and extracted with acetyl acetate (50 ml). The organic layer was dried over MgSO4, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 95:5) to give 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl) )-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanoic acid, NC122 (formula 51) (60 mg, 15%).<*>H NMR 8 7.98 (d, 1H, J= 1.8 Hz), 7.97 (d, 1H, J= 8.3 Hz), 7.86 (dd, 1H, J = 8.3, 1.8 Hz), 7 .42 (d, 2H, J= 8.5 Hz), 7.24 (d, 2H, J= 8.5 Hz), 2.79 (t, 2H, J= 7.5 Hz), 2.62 -2.68 (m, 2H), 2.51-2.59 (m, 2H), 2.47 (t, 2H, J= 7.5 Hz), 2.14-2.26 (m, 1H ), 2.06 (p, 2H, J= 7.5 Hz), 1.60-1.70 (m, 1H).

A solution of DMSO (0.01 mL, 0.12 mmol) in dry dichloromethane (1 mL) was added to a stirred solution of oxalyl chloride (0.01 mL, 0.09 mmol) in dry dichloromethane (2 mL) at - 78 °C. After 15 min. became a dichloromethane solution of 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3,4]octan-5-yl)phenyl )butanamide, NC47 (Formula 52) (35 mg, 0.07 mmol) added to the reaction mixture. Stirring was continued for 20 min. at -78 °C, and then triethylamine (0.03 mL, 0.22 mmol) was added. After 30 min. at -78 °C, the reaction mixture was warmed to room temperature, and then the reaction was quenched with saturated NH 2 Cl solution. The reaction mixture was diluted with dichloromethane and extracted with dichloromethane. The organic layer was dried over MgSO 4 , concentrated and chromatographed (dichloromethane:acetone, 95:5) to give 4-(5-(4-(3-cyanopropyl)phenyl)-8-oxo-6-thioxo-5,7- diazaspiro[3.4]octan-7-yl)-2-(trifluoromethyl)benzonitrile, NC57 (Formula 53)

(29 mg, 87%) as a viscous oil.<*>H NMR 6 7.98 (d, 1H, J= 1.8 Hz), 7.98 (d, 1H, J= 8.3 Hz), 7.86 (dd, 1H, J= 8.3, 1.8 Hz), 7.43 (d, 2H, J= 8.4 Hz), 7.27 (d, 2H, J= 8.4 Hz ), 2.90 (t, 2H, J= 7.3 Hz), 2.63-2.73 (m, 2H), 2.52-2.62 (m, 2H), 2.42 (t, 2H, J= 7.3 Hz), 2.18-2.30 (m, 1H), 2.07 (p, 2H, J= 7.3 Hz), 1.63-1.73 (m, 1H ).

One skilled in the art can modify and/or combine the syntheses described herein to make other diarylhydantoin compounds.

Pharmacological examination of the compounds

Compounds for which synthetic routes are described above were identified by screening hormone-refractory prostate cancer cells for antagonistic and agonistic activities against AR using screening procedures similar to those in PCT applications US04/42221 and US05/05529. A number of compounds showed potent antagonistic activities with minimal agonistic activities for overexpressed AR in hormone-refractory prostate cancer.

Li vitro biological test

Effect of compounds on AR in a reporter assay

The compounds were subjected to tests using an artificial AR response reporter system in a hormone-refractory prostate cancer cell line. In this system, the prostate cancer LNCaP cells were engineered to stably express approximately 5-fold higher levels of AR than the endogenous level. Exogenous AR has similar properties to endogenous AR in that both are stabilized by a synthetic androgen RI 881. The AR overexpressing cells were also engineered to stably incorporate an AR response reporter, and the reporter activity of these cells shows features of hormone-refractory prostate cancer. It responds to a low concentration of a synthetic androgen RI 881, is inhibited only by high concentrations of bicalutamide (see Table 1) and shows agonistic activity with bicalutamide (Figure 1 and Table 2). Consistent with published data, bicalutamide inhibited the AR response reporter and did not have agonistic activity in hormone-sensitive prostate cancer cells (Figure 2).

Table 1

We examined the antagonistic activity of the compounds whose syntheses are described above in the presence of 100 pM of RI 881. Engineered LNCaP cells (LNCaP-AR, also abbreviated LN-AR) were maintained in Iscove's medium containing 10% fetal bovine serum (FBS). Two days before drug treatment, the cells were cultured in Iscove's medium containing 10% charcoal-stripped FBS (CS-FBS) to remove androgens. The cells were split and cultured in Iscove's medium containing 10% CS-FBS with 100 µM of RI 881 and increasing concentrations of test compounds. After two days of incubation, reporter activities were tested.

Table 1 lists the IC50 of the compounds to inhibit AR in hormone-refractory prostate cancer. The control substance bicalutamide has an IC50 of 889 nM. Most of the compounds identified (diarylthiohydantoins) have IC50 between 100 to 200 nM in inhibiting AR in hormone-refractory prostate cancer. In contrast, antiandrogenic compounds listed as examples in US Patent No. 5,705,654, such as reference examples 30-2, 30-3, 31-2, 31-3 and 24-3 (NC24-NC28) have no inhibitory activities on AR in this system.

Antagonistic activities against AR in hormone-refractory prostate cancer measured by an AR response reporter and by endogenous PSA expression are given below for reference.

Table 2

A previously unrecognized feature of AR overexpression in hormone-refractory prostate cancer is the ability to switch antagonists to agonists. Therefore, only those compounds with minimal or no agonistic activities are qualified to be antiandrogens for this disease. To determine the agonistic activities of different compounds, we examined their stimulatory activities on the AR using the AR response reporter as the target in the LN-AR system in the absence of RI 881. Table 2 lists the agonistic activities of different compounds. Consistent with previous results, bicalutamide activated AR in hormone-refractory prostate cancer. The diarylthiohydantoin derivatives, such as reference examples 7-3b (NC7), 33 (NC34), 34 (NC35) and 35 (NC36) have no agonistic activity. In contrast, RU59063 and other antiandrogenic compounds listed as examples in US Patent No. 5,705,654, such as reference examples 30-2, 30-3, 31-2, 31-3 and 24-3 (NC24-NC28), strongly activated AR in hormone-refractory prostate cancer.

Agonistic activities of selective test substances on AR response reporter in hormone-refractory prostate cancer are given below for reference.

To investigate the specificity of AR inhibitors, selective compounds were tested in LNCaP cells with an overexpression of glucocorticoid receptor (GR), the closest member of AR in the nuclear receptor family. These cells also carry a GR response reporter, and the reporter activity was induced by dexamethasone, a GR agonist, and the induction was blocked by RU486, a GR inhibitor. Reference Example 7-3b (NC7) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) had no effect on GR in this system.

Effect of compounds on AR by measuring secreted levels of prostate-specific antigen (PSA)

It is well established that PSA levels are indicators of AR activity in prostate cancer. To investigate whether the compounds affect AR function in a physiological setting, we determined secreted levels of endogenous PSA induced by RI 881 in the AR-overexpressing LNCaP cells (LNCaP-AR, also abbreviated LN-AR). The LNCaP-AR cells are a line of lymph node carcinoma of the prostate cells transduced with a plasmid expressing androgen receptors. LNCaP-AR cells were maintained in Iscove's medium containing 10% FBS. Two days before drug treatment, the cells were cultured in Iscove's medium containing 10% CS-FBS to remove androgens. The cells were split and cultured in Iscove's medium containing 10% CS-FBS with appropriate concentrations of RI 881 and the test compounds. After four days of incubation, secreted PSA levels were tested using a PSA ELISA kit (American Qualex, San Clemente,

ABOUT).

The secreted PSA level of LNCaP-AR cells was strongly induced at 25 pM of RI 881. In contrast, PSA was not induced in the LNCaP parent cells until the concentration of RI 881 reached 100 pM. This is consistent with our previous report that AR in hormone-refractory prostate cancer is hypersensitive to androgens. A dose-dependent inhibition of AR activity was performed to determine the IC50s of various compounds in inhibiting PSA expression, and the results are listed in Table 1. The IC50s of the selective compounds on PSA expression strongly resemble those measured by the reporter assay, confirming that the diarylhydantoin derivatives are strong inhibitors of AR in hormone-refractory prostate cancer.

We also investigated agonistic activities of selective compounds on AR in hormone-refractory prostate cancer using secreted PSA as the surrogate marker. To do this, androgen-starved AR overexpressing LNCaP cells were incubated with increasing concentrations of the compounds, for which a synthesis is described above in the absence of RI 881, and secreted PSA in the culture medium was measured 4 days later.

Table 3 lists the agonistic activities of the selective compounds. Consistent with the results obtained from the reporter test has

the diarylthiohydantoin derivatives such as reference examples 7-3b (NC7), 33 (NC34), 34 (NC35), and 35 (NC36) no agonistic activities. In contrast, RU59063 and other antiandrogenic compounds listed as examples in US Patent No. 5,705,654, such as reference examples 30-2 (NC24), 30-3 (NC25) and 31-2 (NC26) stimulated PSA expression in hormone-refractory prostate cancer.

Table 3

Agonistic activities of selective test substances on endogenous PSA in hormone-refractory prostate cancer are given below for reference.

Number of times induction at increasing concentrations of compounds

Effect of compounds on AR mitochondrial activity by MTS test

LNCaP-AR cells were maintained in Iscove's medium containing 10% FBS. The compounds were examined for their effect on the growth of hormone-refractory prostate cancer cells. Overexpressing LNCaP cells were used because these cells behave like hormone-refractory prostate cancer cells in vitro and in vivo (1). We measured mitochondrial activity by the MTS test, a surrogate for growth. LNCaP cells with overexpressed AR (LN-AR) were maintained in Iscove's medium containing 10% FBS. Two days before drug treatment, the cells were cultured in Iscove's medium containing 10% CF-FBS to remove androgens. The cells were then split and cultured in Iscove's medium containing 10% CS-FBS with appropriate concentrations of R1881 and increasing concentrations of the test compounds. After 4 days of incubation, cell growth was monitored by MTS (Promega, Madison, WI).

Consistent with the reporter test and PSA test, growth of AR overexpressing LNCaP was stimulated at 25 microM of RI 881, but the mother cells were not stimulated until RI 881 concentration reached 100 microM. Figure 2 shows the inhibitory effect of selected compounds on the growth of hormone-refractory prostate cancer in the presence of 100 pM of RI 881. The current clinical drug bicalutamide did not inhibit hormone-refractory prostate cancer. In contrast, Reference Example 5-3b (NC2) (4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile) and Reference Example 7 inhibited -3b (NC7) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3,4]oct-7-yl)-2-trifluoromethylbenzonitrile) hormone-refractory prostate cancer with high potency.

We investigated whether growth inhibition in the MTS test occurs when targeting AR, reference example 5-3b (NC2) (4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl] -2-trifluoromethyl-benzonitrile) and reference example 7-3b (NC7)

(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3,4]oct-7-yl)-2-trifluoromethylbenzonitrile) was tested in DU-145 cells, a prostate cancer cell line lacking AR expression. These compounds had no growth inhibitory effect on DU-145 cells. The compounds did not inhibit cells other than AR-expressing prostate cancer cells, as they had no growth effect on MCF7 and SkBr3, two commonly used breast cancer cells, or 3T3, a normal mouse fibroblast cell line.

Examples of in vitro biological activity of diarylthiohydantoin derivatives are shown in Figures 3 and 4. Based on relative luciferase activity, e.g. fig.3 that at a concentration of 500 nM the compounds are ranked in order of most active to least active as follows: NC67 > NC68 > NC66 > NC69 > NC77 = NC53 > bicalutamide. Based on the relative PSA level, it was e.g. found that at a concentration of 500 nM, the compounds are ranked in order of most active to least active as follows: NC50 > NC48 > NC7 > NC43 > NC44 > NC49 > NC50 > NC45 > bicalutamide. Based on e.g. relative MTS units indicate fig. 4 that at a concentration of 500 nM the compounds are ranked in order of most active to least active as follows: NC70 > NC7 > NC122 > NC53 > bicalutamide.

Inhibitory effect on hormone-refractory and hormone-sensitive prostate cancer xenograft tumors

Compounds of the invention have been used to investigate whether the diarylhydantoin derivatives have in vivo effects on hormone-refractory prostate cancer. First, we examined this connection on xenograft tumors established from AR-overexpressing LNCaP cells. Engineered cells in Matrigel (Collaborative Biomedical) are injected subcutaneously into the flanks of castrated male SOD mice. Tumor size is measured weekly in three dimensions by using calibration targets. After xenograft tumors have established

(tumor size of at least 40 mm<3>) mice with tumors are randomized and treated with different doses of compounds orally once daily. The inhibitory effect on growth of AR overexpressing LNCaP xenograft model is studied as follows. Mice with established LN-AR xenograft tumors are randomized and treated with indicated compounds orally once daily. Tumor size is measured by calibration targets.

Consistent with clinical observation, current clinical drug bicalutamide did not inhibit growth of hormone-refractory prostate cancer (same as delivery agent). In contrast, compounds according to the invention inhibited the growth of these tumors, and the inhibition is dose-dependent. Furthermore, the compounds inhibited PSA expression, the clinical marker of hormone-refractory prostate cancer.

Compounds of the invention have also been tested in another xenograft model of hormone-refractory prostate cancer, hormone-refractory LAPC4. This model was established from passage of hormone-sensitive prostate cancer in castrated mice, which mimics the clinical progression of prostate cancer (2). Similar to the findings using AR overexpressing LNCaP xenograft model, current clinical drug bicalutamide did not inhibit growth and PSA expression in hormone-refractory LAPC4 xenograft model (same as delivery agent). In contrast, compounds of the invention inhibited growth and PSA expression of these tumors.

Figure 6 presents the results of an experiment where cells from the LNCaP hormone-sensitive model were xenografted into mice (106 cells of LNCaP were injected into mice). A first set of mice was treated with NC53, a second set of mice was treated with Casodex, and a third set of mice was treated with delivery vehicle solution. Each set included 6 mice. The mice were treated with 10 mg/kg per day. Fig. 6 presents the results as a graph of tumor volume as a function of time. Mice treated with delivery vehicle solution as a control showed the fastest increase in tumor volume. Mice treated with Casodex and mice treated with NC53 showed similar rates of tumor growth, slower than mice treated with delivery vehicle solution.

Inhibitory effect on the growth of hormone-sensitive prostate cancer cells

To determine whether the diarylthiohydantoin derivatives also inhibited hormone-sensitive prostate cancer cells, we tested some selective compounds on the growth of LNCaP cells by measuring the MTS of mitochondrial activities. Androgen-starved LNCaP cells are treated with increasing concentrations of DMSO as delivery agent or test substances in the presence of 1 pM of RI 881. After 4 days of incubation, cell growth is measured by the MTS test. Compounds of the invention inhibit hormone-sensitive prostate cancer with a higher potency than bicalutamide.

In vivo biological test

All animal experiments were performed in accordance with the guidelines of the Animal Research Committee of the University of California, Los Angeles. Animals were purchased from Taconic and maintained in a laminar flow tower in a colony with defined flora. LNCaP-AR and

LNCaP vector cells were maintained in RPMI medium supplemented with 10% FBS. 106 cells in 100 µl of 1:1 Matrigel to RPMI medium were injected subcutaneously into the flanks of intact or castrated male SOD mice. Tumor size was measured weekly in three dimensions (length x width x depth) using calibration measures. Mice were randomized to treatment groups when tumor size reached approximately 100 mm3. Drugs were given orally every day at 10 mg/kg and 50 mg/kg. To obtain pharmacodynamic results, animals were imaged via an optical CCD camera 3 hours after the last dose of treatment. An ROI is drawn over the tumor for luciferase activity measurement in photons/second. The right panels were a representation of the ROI measurements. Data are shown in Figures 7 and 8. Over 18 days, NC53 was effective in preventing tumor growth and even causing tumor shrinkage, and was clearly more effective than bicalutamide.

The pharmacokinetics of bicalutamide 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-toluene [NC7], iV- methyl 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanamide [NC48] and N-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluorobenzamide ( 52d) [NC53] was evaluated in vivo using 8-week-old FVB mice purchased from Charles River Laboratories. Mice were divided into groups of three for each time point. Two mice were not treated with drug, and two other mice were treated with delivery vehicle solution. Each group was treated with 10 mg per kilograms of body weight.

The drug was dissolved in a mixture 1:5:14 of DMSO : PEG400 : H 2 O (delivery solution) and was administered to mice through the tail vein. The animals were warmed under a heat lamp for approximately 20 minutes before treatment to dilate their tail veins. Each mouse was placed in a mouse restraint apparatus (Fisher Sei. Cat# 01-288-32A) and injected with 200 µl of drug in delivery vehicle solution into the distended tail vein. After drug administration, animals were euthanized via CO2 inhalation at various time points: 5 min., 30 min., 2 h, 6 h, 16 h. Animals were immediately bled after exposure to CO2 via cardiac puncture (1 ml BD syringe + 27G 5/8 needle). For oral dosing, the drug was dissolved in a mixture 50:10:1:989 of DMSO : carboxymethylcellulose : Tween80:H2O before oral administration via a pre-filled syringe.

The serum samples were analyzed to determine drug concentration by HPLC (Waters 600 pump, Waters 600 controller and Waters 2487 detector) equipped with an Alltima Cl 8 column (3(x, 150 mmx4.6 mm). The NC7, NC48 and NC53 compounds was detected at 254 nm wavelength, and bicalutamide was detected at 270 nm wavelength.

The samples for HPLC analysis were prepared according to the following procedure:

- Blood cells were separated from serum by centrifugation.

- To 400 µl of serum were added 80 µl of a 10 µM solution of an internal standard and 520 µl of acetonitrile. Precipitation occurred.

- The mixture was vortexed for 3 minutes and then placed under ultrasound for 30 minutes.

- The solid particles were filtered off and separated by centrifugation.

- The filtrate was dried under an argon stream to dryness. The sample was readjusted to 80 µl with acetonitrile before analysis by HPLC to determine drug concentration.

- Standard curve of drug was used to improve precision.

The concentration of NC53 in plasma as a function of time resulting from intravenous and from oral administration is shown in Figure 9. The steady state concentration (Css) of bicalutamide, NC48 and NC53 is shown in Table 4. The steady state concentration of NC53 is mainly as as good as that of bicalutamide, and significantly better than NC48. Androgen receptor activity may encompass several aspects of stimulation and inhibition of androgen receptor behavior, including but not limited to the following: inhibitory concentration (IC50) in an AR response reporter system or a prostate-specific antigen-secreting system; fold induction associated with increasing concentrations in an AR response reporter system or a prostate-specific antigen-secreting system; associated tumor growth in an animal; the binding affinity of an androgen receptor to a compound; androgen receptor recruitment to a prostate-specific antigen enhancer or a prostate-specific antigen promoter; androgen receptor nuclear translocation; and destabilization of an androgen receptor protein.

In vitro tests

Figure 10 presents the relative binding affinities of compounds for the ligand binding domains of rat androgen receptor (rat AR) as determined with competitor test kits (invitrogen). Fluorescence polarization was used as a result. Each hormone dose was performed in triplicate, and the relative error was determined by calculating the standard error of the three values from the mean. The study controlled for minimal competition (delivery agent alone), no receptor, no fluorescent ligand, and maximal competition (10"<5>MR1881, progesterone, E2, or dexamethasone). Curves were fitted using a single binding site competition model (Prism statistical analysis software package was used). RI 881 had the lowest equilibrium dissociation constant, Ki = 4 nM (and therefore the rat androgen receptor had the highest affinity for RI 881 of the four compounds tested). RU59063 had an equilibrium dissociation constant of Ki = 20 nM, and NC53 had an equilibrium dissociation constant of Ki = 0.8 µM Casodex had an equilibrium dissociation constant of Ki = 0.4 µM (and therefore the rat androgen receptor had the lowest affinity for Casodex of the four compounds tested).

NC53 and Casodex had similar equilibrium dissociation constants, and therefore the rat androgen receptor had a similar affinity for these compounds.

NC53 prevented androgen receptor (AR) recruitment and RNA polymerase II (Pol IT) recruitment to the PSA enhancer and to the PSA promoter. Figure 11 presents the results of the study. Materials used were Chromatin IP with AR (Upstate, cat# 06-680) and Pol II (Covance, cat# MMS-126R). LNCaP (ATCC) cells were plated in full serum. On the day of the experiment, the plate was washed once with 1x PBS, and 5% CSS was added for 3 days. For a first set of experiments, 10 µM of NC53 was added (R), for a second set of experiments, 10 µM of bicalutamide (C) was added, and for a third set of experiments, 1 nM of RI 881 was added (+). Each of these compounds was added for 6 hours. In a fourth set of experiments, a control, no additional compound was added (-). 6 hour time points were run at 28 cycles. ChIP kit from Upstate (cat# 17-295) was used. Enhancer and promoter primers were obtained from Louie (PNAS 2003 Vol. 100, pp. 2226-2230) and Shang (Molecular Cell 2002 vol. 9, pp. 601-610), respectively. The darker image for experiments where NC53 (R) was added indicated that NC53 blocked androgen receptor and prevented RNA polymerase II from forming a transcriptional complex on the prostate-specific antigen (PSA) gene. In contrast, the brighter imaging for experiments where bicalutamide (Casodex, C) was added indicated that in the presence of bicalutamide, androgen receptor and RNA polymerase II were still recruited to the PSA elements to transcribe PSA mRNA.

NC53 inhibited androgen receptor nuclear translocation in LNCaP cells. Figures 12 and 13 present the results of the study. LNCaP cells were plated in 5% CSS. A first set of cells was treated with 10 µM NC53 (R), a second set of cells was treated with 10 µM bicalutamide (C), and a third set of cells was treated with 1 nM RI 881 (+). A fourth set of cells served as a control (−). TOPOI (Santa Cruz, cat# sc-32736) was used to control nuclear fraction, and GAPDH (Santa Cruz, cat# sc-20357) was used to control cytoplasmic fraction. LNCaP cells were harvested for subcellular fractionation or stained with a FITC (Santa Cruz) labeled anti-androgen receptor (AR) antibody (Santa Cruz, cat# sc-815). From the subcellular fractionation, images were obtained, as shown in Fig. 12. The darker imaging in the nuclear fraction for the bicalutamide (Casodex, C) treated sample indicated that bicalutamide induced androgen receptor nuclear translocation. The bright image for the NC53 (R) treated sample indicated that NC53 abrogated nuclear translocation. For the AR-FITC test, coverslips were mounted on glass slides using DAPI-containing medium, and cells were imaged 24 hours later using a fluorescence Nikon microscope at X60 with filters for DAPI and FITC. In the IAR-FITC test, the nuclei of the RI881 and of the bicalutamide-treated cells became clearly green, as shown in fig. 13, indicating that nuclear translocation of the androgen receptor occurred. In contrast, the nuclei of the DMSO and of the NC53 treated cells were less green.

NC53 destabilized androgen receptor proteins in LNCaP cells. Figure 14 shows the results of the study. The study was performed by plating IO5 LNCaP (fgc) cells in 5% CSS for 3 days. 100 µM of RI 881 was added to a first set of cells (+), 10 µM of bicalutamide was added to a second set of cells (B), 10 µM of NC53 was added to a third set of cells (RD), 100 pM of R1881 and 10 µM of bicalutamide were added to a fourth set of cells (B+), and 100 µM of R1881 and 10 µM of NC53 were added to a fifth set of cells (RD+). Neither RI 881, bicalutamide nor NC53 was added to a sixth set of cells (-). The cells were allowed to remain with added bicalutamide, NC53 and/or RI 881 for 24 hours (or in the case of the (-) kit, without any of these for 24 hours). In fig. 14, the dark imaging for the kit to which bicalutamide (B) was added, and for the kit to which bicalutamide and RI 881 (B+) were added, indicated that the androgen receptor proteins were upregulated when these combinations of compounds were added. In contrast, the bright imaging for the kit to which NC53 (RD) was added and for the kit to which NC53 and RI 881 (RD+) were added indicated that the addition of NC53 resulted in the degradation of androgen receptor proteins, whether RI 881 was present or not.

Ranking of compounds in ranks

Tables 5-10 present diarylhydantoin compounds grouped in rows 1-6. Table 11 presents diarylhydantoin compounds that have not been placed in a series. The placement of compounds in ranks was based on available data coupled with analytical judgement. Data considered included in vitro tests (AR response reporter system in LNCaP cell line, PSA level measurement, MTS mitochondrial test) and in vivo experiments (tumor size measured directly or by emission induced by luciferase reporter gene, pharmacokinetic tests based on blood plasma levels). Not every compound was subjected to every test. Not all data that was generated is shown. Rating was used to rank compounds relative to each other for their utility in the treatment of prostate cancer, particularly when ranking two compounds for which the same experiments were not performed. Properties considered in establishing the ranking included AR antagonism activity, lack of AR agonism in hormone-refractory cells, prevention of tumor growth, tumor shrinkage, and pharmacokinetic behavior, where a longer residence time in blood is beneficial.

Row 1

In general, series 1 compounds are diarylhydantoins with a disubstituted left-handed aryl ring that is disubstituted on the right hydantoin carbon, and has either an oxygen or N substituent on the left hydantoin carbon. It is expected that the amido substituent hydrolyzes to an oxygen in aqueous solutions such as encountered in biological systems in vitro and in vivo. NC63 has good activity with an iodine instead of a CF3 substituent on the left-handed aryl ring.

Series 1 compounds (see Table 5) were judged to be much better than bicalutamide for the treatment of prostate cancer. However, NC7 and NC48 were found to be rapidly metabolized, meaning they have a shorter residence time in blood. NC53 had desirable pharmacokinetics. Figure 16 shows that during treatment with bicalutamide, PSA levels for LNCaP cells remained the same or increased relative to treatment with delivery agent solution, while during treatment with NC53 the PSA level was reduced. Figure 17 illustrates that during treatment with delivery agent solution, tumors continued to increase in size. During treatment with NC53 at a dose of 1 mg per kg body weight per day, in contrast, the rate of tumor growth was reduced, and the size of the tumor proved to be stabilized after about 17 days. During treatment with NC53 at a dose of 10 mg per kg body weight per day, tumor size decreased with time. Figure 18 illustrates that during treatment with NC53 at a dose of 10 mg per kg body weight per day, photon emission associated with luciferase activity was reduced. Figure 19 shows that treatment with NC53 at this dose resulted in a reduction or stabilization of tumor size and a reduction in photon emission associated with luciferase activity. Figure 20 shows that upon treatment with NC53, NC54, NC55, NC56 and NC57 at doses of 100, 200, 500 and 1,000 nM, PSA levels of LN-AR cells were reduced. The higher the dose, the lower the PSA level. Figure 22 presents urogenital tract weight and rate of photon emission associated with luciferase activity initially and after 14 days of treatment with bicalutamide or with NC53 for intact and castrated mice. The weight and rate of photon emission increased for both intact and castrated mice. Treatment of castrated mice with NC53 resulted in a reduction in weight and photon emission relative to untreated castrated mice, as did treatment with bicalutamide.

Series 1 compounds are therefore particularly advantageous for use as AR antagonists and as therapeutic agents for hormone-refractory prostate cancer. They may be useful in treating other AR-related diseases or conditions such as benign prostatic hyperplasia, hair loss, and acne. These and related compounds may also be useful as modulators of other nuclear receptors, such as glucocorticoid receptor, estrogen receptor, and peroxisome proliferator-activated receptor, and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease, and metabolism-related diseases. They can be useful in tests such as standards or as intermediates or prodrugs.

In table 5, the compounds of the invention are marked with <*>. Other compounds for reference.

Row 2

A number of 2 compounds (see table 6) were significantly better than bicalutamide for the treatment of prostate cancer, although there were indications that NC12 may act as an agonist.

Figure 3 illustrates that compounds NC66, NC67, NC68, NC53 and NC69 in line 1 and NC77 in line 2 dosed at concentrations varying from 124 nM to 1,000 nM acted to reduce luciferase activity in LNCaP-AR cells, while control solutions of DMSO and of bicalutamide had little or no effect. It was found that at concentrations of 1,000 nM compounds NC7 and NC48 in line 1 caused a greater reduction in PSA level of LNCaP-AR cells than NC43, NC44 and NC50 in line 2. Figure 7 presents tumor volume over time, and illustrates that under treatment with bicalutamide or delivery vehicle solution, tumors continued to grow, whereas during treatment with NC53 in row 1, tumors were reduced in size. Figure 8 illustrates that photon emission associated with luciferase activity remained about the same or increased during treatment with bicalutamide relative to treatment with delivery vehicle solution, while photon emission was reduced during treatment with NC53. Figure 23 illustrates that during treatment with bicalutamide there was little or no reduction in PSA levels, while during treatment with NC48 and NC53, PSA levels were reduced. Figure 24 illustrates that the IC50 for NC7, NC48 and NC53 in row 1 was much lower than the IC50 for bicalutamide.

In general, series 2 compounds are structurally similar to series 1 compounds, but with different substituents on the right-handed aryl ring. A number of 2 compounds are advantageous for use as AR antagonists and as therapeutic agents for hormone-refractory prostate cancer. They may be helpful in treating other AR-related disorders or conditions such as benign prostatic hyperplasia, hair loss, and acne. These and related compounds may also be useful as modulators of other nuclear receptors, such as estrogen receptor and peroxisome proliferator-activated receptor and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease and metabolism-related diseases. They can be useful in tests such as standards or as intermediates or prodrugs.

Connections shown below in Table 6 are for reference.

Row 3

A number of 3 compounds (see Table 7) were judged to be slightly better than bicalutamide for the treatment of prostate cancer. NC43, NC44 and NC50 (in row 2) caused a greater decrease in PSA level of LNCaP-AR cells. All of these compounds caused a greater reduction in PSA levels than bicalutamide.

Another series of 3 compounds (not shown) were not diarylthiohydantoins, and were comparable in activity to previously known monoarylhydantoin compounds NC83, NC79 and NC80.

Therefore, series 3 compounds are useful as AR antagonists and as therapeutic agents for hormone-refractory prostate cancer. They may be useful in treating other AR-related diseases or conditions such as benign prostatic hyperplasia, hair loss, and acne. These and related compounds may also be useful as modulators of other nuclear receptors, such as estrogen receptor and peroxisome proliferator-activated receptor and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease and metabolism-related diseases. They can be useful in tests such as standards or as intermediates or prodrugs.

Connections shown below in Table 7 are for reference.

Row 4

A number of 4 compounds (see Table 8) were judged not to be better than bicalutamide for the treatment of prostate cancer. Series 4 NC93 and NC94 and series 1 NC7 are e.g. differing only in the substituent on the lower right carbon of the hydantoin ring. The substituents on the right-handed aryl ring can also affect activity.

Some Series 4 compounds (including those shown and others not shown) were not diaryl compounds (lacking the right-handed aryl ring), were not thiohydantoins, were not disubstituted on the lower right carbon of the hydantoin ring, and/or had substituents other than oxygen or amido on the lower left-hand carbon of the hydantoin ring. This provides evidence for the surprising advantages of diarylthiohydantoins that are disubstituted on the lower right-hand carbon of the hydantoin ring and have oxygen or amido on the lower left-hand carbon of the hydantoin ring.

A number of 4 compounds can therefore be useful as AR antagonists, and as therapeutic agents for hormone-refractory prostate cancer, at least to the extent that they are comparable to bicalutamide. They may be useful in treating other AR-related diseases or conditions such as benign prostatic hyperplasia, hair loss, and acne. These and related compounds may also be useful as modulators of other nuclear receptors, such as estrogen receptor and peroxisome proliferator-activated receptor and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease and metabolism-related diseases. They can be useful in tests such as standards or as intermediates or prodrugs.

Connections shown below in Table 8 are for reference.

Row 5

A number of 5 compounds (see Table 9) were inactive or almost inactive and were therefore worse than bicalutamide for the treatment of prostate cancer. The substituents on the right-handed aryl ring are important in determining activity.

Some series 5 compounds (some of which are shown and some of which are not shown) were not diaryl compounds (lacking the right-handed aryl ring), were not thiohydantoins, were not disubstituted on the carbon on the lower right side of the hydantoin ring, and/or had substituents other than oxygen or amido on the lower left-hand carbon of the hydantoin ring. This provides evidence for the surprising advantages of diarylthiohydantoins that are disubstituted on the lower right-hand carbon of the hydantoin ring and have oxygen or amido on the lower left-hand carbon of the hydantoin ring. In particular, the terminal substituent in NC103, NC104 and NC106 (CEfeNRxRy, where Rx,y = H or methyl) is not seen to contribute to activity in these compounds.

Series 5 compounds would not be desirable for the treatment of prostate cancer or as AR antagonists, although these and related compounds may be useful as modulators of other nuclear receptors, such as estrogen receptor and peroxisome proliferator-activated receptor and as therapeutic agents for diseases where nuclear receptors play a role, such as such as breast cancer, ovarian cancer, diabetes, heart disease and metabolism-related diseases. They can be useful in tests such as standards or as intermediates or prodrugs.

Connections shown below in Table 9 are for reference.

Row 6

A number of 6 compounds (see table 10) were inactive or almost inactive and were furthermore strong agonists, and therefore they were much worse than bicalutamide for the treatment of prostate cancer. The comparative compounds were ranked very poorly relative to the invented compounds. Remarkably, NC107 had very poor activity with a chlorine substituent on the left-handed aryl ring, while NC2 with a trifluoromethane and NC63 with iodo ranked in series 1. The results for series 6 compounds provided evidence for the surprising advantages of diarylthiohydantoins disubstituted on the lower right-handed -carbon of the hydantoin ring and has oxygen or amido on the lower left-hand carbon of the hydantoin ring and has certain substituents on the left-hand aryl ring.

Series 6 compounds will not be desirable for the treatment of prostate cancer or as AR antagonists.

Connections shown below in Table 10 are for reference.

Connections not sequenced

For several compounds, there was insufficient experimental data to rank them. These compounds not listed in sequence are presented in table 11.

Based on the data and methods of the invention, and using judgment based on review of many compounds, including some not shown here, one can make some observations about the compounds that are not listed. Comparative example NC108 is expected to be in line 3 with comparative examples NC78-NC80. NC113 is expected to hydrolyse to NC7 (row 1) and should therefore have comparable activity. NC114 is expected to hydrolyze to NC16 (row 1) and should therefore have comparable activity. NC115 is expected to hydrolyze to NC3 (row 1), and NC120 and NC 121 are expected to hydrolyze to NC50 (row 2), and therefore should have comparable activity.

The connections shown below in Table 11 are for reference.

In short, new compounds showing evidence of being far superior to bicalutamide in the treatment of prostate cancer were identified and produced.

Sensitivity of anticancer activity of compounds to structural differences

The inventors have determined that what may turn out to be a small change in the structure of hydantoin compounds can result in a major change in that compound's performance in the treatment of prostate cancer. E.g. NC77 and NC53 differ only by a single fluorine substituent on an aryl ring, and NC53 is in row 1 while NC77 is in row 2, where both are better than bicalutamide for the treatment of prostate cancer, but NC53 is superior. However, NC98, which differs from NC77 only by having an additional carbon atom between the methylcarbamoyl group and the aryl ring, is not better than bicalutamide for the treatment of prostate cancer and is ranked in the 4th rank. The effect of NC77, NC53 and NC98 on luciferase activity can be seen in Figure 25. At a given concentration of compound, the luciferase activity upon exposure to NC77 and NC53 is less than the luciferase activity upon exposure to NC98.

NC4 differs from NC3 only in that an amino group is substituted for a hydroxyl group. While NC3 is in line 1 and is much better than bicalutamide for the treatment of prostate cancer, however, NC4 is in line 4, no better than bicalutamide. The effect of NC3 and NC4 on luciferase activity in the 1AR cell line was studied by administering various compounds at concentrations varying from 1.25 to 10 µmol and observing PSA levels. For a given dose, the luciferase activity upon exposure to NC3 is less than the luciferase activity upon exposure to NC4. The effect of NC3 and NC4 on luciferase activity in the 4AR cell line was studied by administering various compounds at concentrations varying from 1.25 to 10 µmol and observing luciferase activity. For a given dose, the luciferase activity upon exposure to NC3 is less than the luciferase activity upon exposure to NC4. The effect of NC3 and NC4 on PSA levels in the LN/AR cell line was studied by administering various compounds at concentrations varying from 1.25 to 10 µmol and observing luciferase activity. For a given dose, the PSA level upon exposure to NC3 is less than the PSA level upon exposure to NC4.

NC47 and NC48 differ from each other by only a methyl substituent at the end of a carbamoyl group, and both compounds are ranked in series 1, although NC48 has been found to be particularly advantageous. NC46 is the same as NC47, with the exception of a methoxy group substituted for an amino group. However, NC46 is ranked in series 3. NC42 is similar to NC46, but has a smaller carbon in the chain connecting the ester group to the aryl ring; NC42 is ranked number 3. The effect of NC47, NC48, NC42 and NC46 on PSA levels in the LN/AR cell line was studied by administering various compounds at concentrations varying from 125 to 1,000 umol and observing PSA levels. For a given concentration, the PSA level upon exposure to NC47 and NC48 is less than the PSA level upon exposure to NC42 and NC46.

NC68 and NC103 differ from each other in that the former has a methylcarbamoyl group attached to an aryl ring and a dimethyl substituent attached to the thiohydantoin group, while the latter has a methylamino group attached to the right-handed aryl ring and a cyclobutyl substituent attached to the thiohydantoin group. While NC68 is in line 1, much better than bicalutamide for the treatment of prostate cancer, NC103 is in line 5 and is inactive or almost inactive in the treatment of prostate cancer. The effect of NC68 and NC103 on luciferase activity in the LN/AR cell line was studied by administering various compounds at concentrations ranging from 125 to 1,000 µmol and observing luciferase activity. For a given concentration, the luciferase activity upon exposure to NC68 is less than the luciferase activity upon exposure to NC103.

NC16 and NC18 differ from each other in that the substitution of a thio for an oxo group and a dimethyl substituent for a cyclobutyl substituent. While NC16 is in row 1, NC18 is in row 4.

Pharmaceutical compositions and administration

The compounds of the invention are useful as pharmaceutical compositions prepared in a therapeutically effective amount of a compound of the invention, as defined herein, and a pharmaceutically acceptable carrier or diluent.

The diarylhydantoin compound of the invention can be formulated as pharmaceutical compositions and administered to an individual in need of treatment, e.g. a mammal, such as a human patient, in a variety of forms adapted to the chosen route of administration, e.g. orally, nasally, intraperitoneally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into tissue.

Therefore, diarylhydantoin compounds of the invention can be systemically administered, e.g. orally, in combination with a pharmaceutically acceptable delivery vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or inhalation. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets or may be incorporated directly into the food of the patient's diet. For oral therapeutic administration, the diarylhydantoin compounds can be combined with one or more additives and used in the form of ingestible tablets, buccal tablets, drops, capsules, elixirs, suspensions, syrups, wafers and the like. The diarylhydantoin compounds can be combined with an inert carrier in powder form and inhaled by the subject or locked in. Such compositions and preparations should contain at least 0.1% diarylhydantoin compounds. The percentage of the composition and preparations can of course be varied and can conveniently be between about 2% to about 60% of the weight of a given unit dosage form. The amount of diarylhydantoin compounds in such therapeutically useful compositions is such that an effective dosage level will be achieved. The tablets, drops, pills, capsules etc. may also contain the following: binding agents such as gum tragacanth, acacia, corn starch or gelatin; additives such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetener such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen or cherry flavor may be added. When the unit dosage form is a capsule it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. E.g. tablets, pills or capsules can be coated with gelatin, wax, shellac or sugar etc. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methyl and propyl parabens as preservatives, a coloring and flavoring agent such as cherry or orange flavor. Of course, any material used in the manufacture of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts used. In addition, the diarylhydantoin compounds can be incorporated into sustained release preparations and devices. E.g. the diarylhydantoin compounds can be incorporated into time-release capsules, time-release

tablets and time-release pills.

The diarylhydantoin compounds may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the diarylhydantoin compounds can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions, or sterile powders comprising the diarylhydantoin compounds adapted for the immediate preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, liquid and stable under the conditions of manufacture and storage. The liquid carrier or delivery medium may be a solvent or liquid dispersion medium comprising e.g. water, ethanol, a polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycols, etc.), vegetable oils, non-toxic glyceryl esters and suitable mixtures thereof. The correct fluidity can be maintained by e.g. the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be carried out by various antibacterial and antifungal agents, e.g. parabens, chlorobutanol, phenol, sorbic acid, thimerosal etc. In many cases it will be preferred to include isotonic agents, e.g. sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be caused by the formation in the compositions of agents which delay absorption, e.g. aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the diarylhydantoin compounds in the required amount in the appropriate solvent with various of the other ingredients listed above, as necessary, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which provide a powder of the active ingredient plus any additional desired ingredient in the previously sterile filtered solutions.

For topical administration, the diarylhydantoin compounds may be applied in pure form. However, it is generally desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Use of solid carriers includes finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Other solid carriers include non-toxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols or water/alcohol/glycol mixtures, in which the diarylhydantoin compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents may be added to optimize properties for a given application. The resulting liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump type or aerosol sprays. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps and the like. for application directly to the skin of the user.

Examples of useful dermatological compositions that can be used to deliver the diarylhydantoin compounds to the skin are known in the art, see e.g. Jacquet et al. (US Pat. No. 4,608,392), Geria (US Pat. No. 4,992,478), Smith et al. (US Pat. No. 4,559,157) and Wortzman (US Pat. No. 4,820,508).

Useful dosages of the compound of formula I can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice and other animals to humans are known in the field, see e.g. US Pat. No. 4,938,949.

The concentration of the diarylhydantoin compounds in a liquid composition, such as a lotion, can e.g. be from about 0.1-25% by weight or from about 0.5-10% by weight. The concentration of a semi-solid or solid composition such as a gel or a powder may be about 0.1-5% by weight or about 0.5-2.5% by weight.

The amount of the diarylhydantoin compounds required for use in treatment will vary not only with the particular salt selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be subject to the discretion of the attending physician or the clinician.

Effective dosages and routes of administration of agents of the invention are conventional. The exact amount (effective dose) of the agent will vary from individual to individual, depending on e.g. the nature, age, weight and general clinical condition of the individual, the severity or mechanism of any disorder being treated, the particular agent or means of delivery used, the method and schedule of administration, etc. A therapeutically effective dose can be determined empirically by conventional procedures known to those skilled in the art. See e.g. The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. An effective dose can e.g. be estimated initially either in cell culture tests or in suitable animal models. The animal model can also be used to determine the appropriate concentration areas and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans. A therapeutic dose may also be selected by analogy to dosages for comparable therapeutic agents.

The particular route of administration and dosage regimen will be chosen by the treating clinician, taking into account the particulars of the case (eg, the individual, the disease, the level of disease involved, and whether the treatment is prophylactic). Treatment may involve daily or multiple daily doses of the compound(s) over a period of a few days to months, or even years.

In general, however, a suitable dose will be in the range from about 0.001 to about 100 mg/kg, e.g. from about 0.01 to about 100 mg/kg of body weight per day, such as over about 0.1 mg per kilogram or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day. E.g. a suitable dose can be about 1 mg/kg, 10 mg/kg or 50 mg/kg of body weight per day.

The diarylhydantoin compounds are conveniently administered in unit dosage form; e.g. containing 0.05 to 10,000 mg, 0.5 to 10,000 mg, 5 to 1,000 mg or about 100 mg of active ingredient per unit dosage form.

The diarylhydantoin compounds can be administered to achieve peak plasma concentrations of e.g. from about 0.5 to about 75 µM, about 1 to about 50 µM, about 2 to about 30 µM or about 5 to about 25 µM. Exemplary desirable plasma concentrations include at least or no more than 0.25, 0.5, 1.5, 10, 25, 50, 75, 100 or 200 µM. Plasma levels can e.g. be from about 1 to 100 micromolar or from about 10 to about 25 micromolar. This can be achieved by e.g. the intravenous injection of a 0.05 to 5% solution of the diarylhydantoin compounds, optionally in saline or orally administered as a bolus containing about 1-100 mg of the diarylhydantoin compounds. Desirable blood levels can be maintained by continuous infusion to provide about 0.00005 - 5 mg per kg body weight per hour, e.g. at least or not more than 0.00005, 0.0005, 0.005, 0.05, 0.5 or 5 mg/kg/hr. Alternatively, such levels can be achieved by periodically recurring infusions containing about 0.0002 - 20 mg per kg body weight, e.g. at least or not more than 0.0002, 0.002, 0.02, 0.2, 2, 20 or 50 mg of the diarylhydantoin compounds per kg of body weight.

The diarylhydantoin compounds may conveniently be presented in a single dose or as divided doses administered at convenient intervals, e.g. as two, three, four or more underdoses per day. The underdose itself can be further divided, e.g. in a number of separate loosely divided administrations, such as multiple inhalations from a breathing device.

A number of the above-identified compounds show little or no agonistic activity with respect to hormone-refractory prostate cancer cells. Because these compounds are strong AR inhibitors, they can be used not only in the treatment of prostate cancer, but also in the treatment of other AR-related diseases or conditions, such as benign prostatic hyperplasia, hair loss and acne. Because AR belongs to the family of nuclear receptors, these compounds can serve as scaffolds for drug synthesis targeting other nuclear receptors, such as estrogen receptor and peroxisome proliferator-activated receptor. They can therefore be further developed for other diseases such as breast cancer, ovarian cancer, diabetes, heart disease and metabolism-related diseases, where nuclear receptors play a role.

A sequence for the chemical synthesis of compounds described herein is shown below. The cyanohydrins 10bc are converted to the different cyanoamines 12bc by reaction with the different anilines 1b (10b and 1a give 12b, 10c and 11b give 12c). In a separate process, the aniline 13 is converted in one step to the isothiocyanate 14. Addition of 12bc to 14 followed by treatment with mild acid produces the desired thiohydantoins 5 (NC55) and 6 (NC56) in good yield.

The embodiments illustrated and discussed in this disclosure are intended only to demonstrate to those skilled in the art the best way known to the inventors to make and use the invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as understood by those skilled in the art in light of the above demonstrations. It must therefore be understood that within the scope of the claims, the invention can be practiced in a different way than what is specifically described.

Claims (18)

1. Compound, characterized in that it has the formula wherein R 1 and R 2 are, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms; wherein R 3 is cyanoalkyl; and where R 4 is hydrogen or fluorine. 2. Compound according to claim 1, characterized in that Ri and R2 are, together with the carbon to which they are connected, a cycloalkyl group with 4 carbon atoms. 3. Compound, characterized in that it has the formula 4. Compound, characterized in that it has the formula 5. A compound according to any one of claims 1 to 4 for use in the treatment of the human or animal body by therapy. 6. Pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of a compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. 7. Pharmaceutical composition according to claim 6, for use in the treatment of the human body or the animal body in therapy. 8. Pharmaceutical composition according to claim 6, for use in a method for treating a hyperproliferative disorder. 9. Composition according to claim 8, characterized in that a) the composition has a form selected from the group consisting of a solution, dispersion, suspension, powder, capsule, tablet, pill, time-release capsule, time-release tablet, time-release pill; or b) the composition has a form selected from the group consisting of a capsule, tablet and pill; or c) the composition is administered by intravenous injection, by injection into tissue, intraperitoneally, orally or nasally; or d) the composition is administered at a dose of the compound ranging from about 0.001 mg/kg body weight per day to about 100 mg per kilogram body weight per day; or e) the composition is administered at a dose of the compound ranging from about 0.01 mg/kg body weight per day to about 100 mg per kilogram body weight per day; or f) the composition is administered at a dose of the compound ranging from about 0.1 mg/kg body weight per day to about 10 mg per kilogram body weight per day; or g) the composition is administered at a dose of the compound in the range of about 1 mg/kg body weight per day; or h) the composition interferes with transcription of prostate specific antigen mRNA; or i) the composition prevents nuclear translocation of an androgen receptor protein; or j) the composition destabilizes an androgen receptor protein. 10. Use of a pharmaceutical composition according to claim 6 or 9 for the preparation of a medicament for use in a method for treating a hyperproliferative disorder. 11. A method for synthesizing a) the compound of formula characterized in that it comprises mixing NJf-dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile and DMF and heating under reflux to form a first mixture; adding an alcohol and an acid to the first mixture to form a second mixture; reflux of the second mixture; cool the second mixture; combining the second mixture with water and extracting an organic layer; and isolating the compound from the organic layer; or b) the compound of formula characterized in that it comprises mixing DMSO, dichloromethane and oxalyl chloride to form a first mixture, add 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3,4]octan-5-yl)phenyl)butanamide to the first mixture to form a second mixture; adding triethylamine to the second mixture to form a third mixture; heating the third mixture and quenching it with aqueous NH 4 Cl to form a fourth mixture; extracting an organic layer from the fourth mixture; isolate the compound from the organic layer. 12. In vitro method, characterized in that it comprises contacting a mammalian cell capable of expressing prostate specific antigen with a sufficient amount of a diarylthiohydranion compound to interfere with transcription of prostate specific antigen mRNA, wherein the diarylthiohydanion compound has the formula or has the formula wherein R 1 and R 2 are, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms; wherein R 3 is cyanoalkyl; and where R 4 is hydrogen or fluorine. 13. The method according to claim 12, characterized in that a) the diarylthiohydantoin compound is selected from the group consisting of b) the diarylthiohydantoin compound prevents the formation of a transcription complex on a prostate-specific antigenic gene; or c) the diarylthiohydantoin compound prevents an androgen receptor protein from complexing a prostate-specific antigenic gene; or d) the diarylthiohydantoin compound prevents an RNA polymerase JJ from complexing a prostate-specific antigenic gene. 14. In vitro method, characterized in that it comprises contacting a mammalian cell with a sufficient amount of a diarylthiohydranion compound to prevent nuclear translocation of an androgen receptor protein and/or destabilize an androgen receptor protein, wherein the diarylthiohydanion compound has the formula or wherein R 1 and R 2 are, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms; wherein R 3 is cyanoalkyl; and where R 4 is hydrogen or fluorine. 15. Pharmaceutical composition according to claim 8 or use according to claim 10, wherein the hyperproliferative disorder is selected from the group consisting of prostate cancer, hormone-refractory prostate cancer and hormone-sensitive prostate cancer. 16. Pharmaceutical composition according to claim 8 or use according to claim 10, wherein the hyperproliferative disorder is benign prostatic hyperplasia. 17. Pharmaceutical composition according to claim 8 or use according to claim 10, wherein the hyperproliferative disorder is selected from the group consisting of breast cancer and ovarian cancer. 18. Compound according to claim 1, characterized by atlUer hydrogen.